A key factor in the neurodegenerative condition glaucoma is injury to axons in the optic nerve, which triggers subsequent degeneration of Retinal Ganglion Cells (RGCs). Recent work has revealed a key role for Dual Leucine-zipper Kinase (DLK), supported by DLK's close relative Leucine-zipper Kinase (LZK), in RGC degeneration. There is therefore great interest in identifying DLK/LZK inhibitors to protect RGCs in glaucoma and other neurodegenerative conditions. However, current strategies all seek to inhibit DLK/LZK's kinase activity, an approach prone to undesired `off-target' effects due to the large number of similar kinases. We recently found that DLK and LZK, but not homologous kinases, are covalently modified by the lipid palmitate. Focusing on DLK, we have found that this process, palmitoylation, is essential for (i) DLK-dependent signaling and (ii) DLK-dependent degeneration of RGC cell bodies 3 weeks after Optic Nerve Crush (ONC, a partial model of glaucomatous injury) in vivo. These findings suggest that preventing DLK/LZK palmitoylation could be as effective a therapeutic strategy as inhibition of DLK's kinase activity, while potentially minimizing off-target issues. In this proposal we will first extend our preliminary findings to determine whether preventing DLK palmitoylation (by replacement of endogenous DLK with a palmitoyl-site mutant) confers long-lasting protection to RGCs after ONC in vivo. We will also determine whether specific loss of palmitoyl-DLK protects RGC axons proximal to the injury site. In parallel studies we will assess the extent to which ZDHHC17, a Palmitoyl Acyltransferase enzyme that controls DLK palmitoylation in cultured neurons, is essential for ONC-induced RGC degeneration. We will determine whether a novel ZDHHC17 interaction motif in DLK is essential for DLK- ZDHHC17 binding and for RGC degeneration by palmitoyl-DLK (Aim 1). In parallel studies, we will build on a novel High Content Imaging Assay that we have recently optimized in order to screen for inhibitors of DLK palmitoylation. Together with our Co-I Dr. Don Zack we will then determine whether inhibitors from this screen can protect both mouse and human Embryonic Stem Cell-derived RGCs in a model of axonal injury that was recently optimized by the Zack lab (Aim 2). Results from Aim 1 should provide new insights into mechanisms of RGC degeneration, while Aim 2 may yield an entirely new class of neuroprotectants, with broad implications for the treatment of glaucoma and perhaps other neurodegenerative conditions.